Respiratory nitrate reductase alpha subunit

ABSTRACT

The invention provides respiratory nitrate reductase alpha subunit polypeptides and polynucleotides encoding respiratory nitrate reductase alpha subunit polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing respiratory nitrate reductase alpha subunit polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This invention claims benefit to U.S. Provisional Patent Application No.60/086,579, filed May 22, 1998.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the reductases family, aswell as their variants, herein referred to as "respiratory nitratereductase alpha subunit," "respiratory nitrate reductase alpha subunitpolynucleotide(s)," and "respiratory nitrate reductase alpha subunitpolypeptide(s)" as the case may be.

BACKGROUND OF THE INVENTION

It is particularly preferred to employ Staphylococcal genes and geneproducts as targets for the development of antibiotics. TheStaphylococci make up a medically important genera of microbes. They areknown to produce two types of disease, invasive and toxigenic. Invasiveinfections are characterized generally by abscess formation effectingboth skin surfaces and deep tissues. S. aureus is the second leadingcause of bacteremia in cancer patients. Osteomyelitis, septic arthritis,septic thrombophlebitis and acute bacterial endocarditis are alsorelatively common. There are at least three clinical conditionsresulting from the toxigenic properties of Staphylococci. Themanifestation of these diseases result from the actions of exotoxins asopposed to tissue invasion and bacteremia. These conditions include:Staphylococcal food poisoning, scalded skin syndrome and toxic shocksyndrome.

The frequency of Staphylococcus aureus infections has risen dramaticallyin the past few decades. This has been attributed to the emergence ofmultiply antibiotic resistant strains and an increasing population ofpeople with weakened immune systems. It is no longer uncommon to isolateStaphylococcus aureus strains that are resistant to some or all of thestandard antibiotics. This phenomenon has created an unmet medical needand demand for new anti-microbial agents, vaccines, drug screeningmethods, and diagnostic tests for this organism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces "functional genomics," that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on "positional cloning" and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the respiratory nitrate reductase alpha subunit embodiments of theinvention, that have a present benefit of, among other things, beinguseful to screen compounds for antimicrobial activity. Such factors arealso useful to determine their role in pathogenesis of infectiondysfunction and disease. There is also a need for identification andcharacterization of such factors and their antagonists and agonists tofind ways to prevent ameliorate or correct such infection, dysfunctionand disease.

SUMMARY OF THE INVENTION

The present invention relates to respiratory nitrate reductase alphasubunit in particular respiratory nitrate reductase alpha subunitpolypeptides and respiratory nitrate reductase alpha subunitpolynucleotides, recombinant materials and methods for their production.In another aspect, the invention relates to methods for using suchpolypeptides and polynucleotides, including treatment of microbialdiseases, amongst others. In a further aspect, the invention relates tomethods for identifying agonists and antagonists using the materialsprovided by the invention, and for treating microbial infections andconditions associated with such infections with the identified agonistor antagonist compounds. In a still further aspect, the inventionrelates to diagnostic assays for detecting diseases associated withmicrobial infections and conditions associated with such infections,such as assays for detecting respiratory nitrate reductase alpha subunitexpression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to respiratory nitrate reductase alpha subunitpolypeptides and polynucleotides as described in greater detail below.In particular, the invention relates to polypeptides and polynucleotidesof a respiratory nitrate reductase alpha subunit of Staphylococcusaureus, that is related by amino acid sequence homology to NarG[Staphylococcus carnosus] polypeptide. The invention relates especiallyto respiratory nitrate reductase alpha subunit having a nucleotide andamino acid sequences set out in Table 1 as SEQ ID NO:1 or 3 and SEQ IDNO:2 respectively. Note that sequences recited in the Sequence Listingbelow as "DNA" represent an exemplification of the invention, sincethose of ordinary skill will recognize that such sequences can beusefully employed in polynucleotides in general, includingribopolynucleotides.

                                      TABLE 1                                     __________________________________________________________________________    Respiratory nitrate reductase alpha subunit Polynucleotide and                 Polypeptide Sequences                                                        __________________________________________________________________________    (A) Staphylococcus aureus respiratory nitrate reductase alpha                  subunit polynucleotide sequence [SEQ ID NO:1].                                5' -ctgcaggaattcccaaatgatacatcgaatggcacatatcattcgtcgcaatatgattac               - tatgatgcatttattaagcagcaagaaaatgtaacatatatttcaaccgatcgtgcagat                - gctaatacagtgttatgtcactaatttataaaaaataaatgaataagtaaggtttcaacc                - gagagaatatattcgtgttgaagccttatttgtcgttgtgccaaatttgaacgatttaga                - ttggcaataagcacgaccatacatgattgtgtattatttcaatgaaatcccgttattgat                - agggaaattccttaagtaattaggtgattccctaatattttatatttgtgaaaaaatgta                - caatctaattaaagcaatagtcttgggcattttaaagtatttaaaaacacgacatctaca                - catcgtatttttatacgtaggaggatataaatatgggaaaatttggattgaatttcttta                - agccaacagaaaaatttaatgggaattggtcgatcctagaaagtaaaagtagagaatggg                - aaaaaatgtacagagaacgttggagccacgataaagaagtaagaacaacacatggtgtta                - actgtacaggctcatgttcttggaaagtatttgtgaaaaatggtgtgattaactgggaaa                - atcaacaaactgactatccaagttgtggtccggatatgcctgaatatgaaccgagaggat                - gtccacgaggtgcgtcattctcttggtatgaatacagtccgcttcgaatcaaatatccat                - atattcgtggaaaactctgggatttatggactgaagcattagaagaaaactatggtaatc                - gcgttgctgcatgggcgtctattgttgaaaatgaagacaaagccaaacaatataagcaag                - cccgaggtatgggagggcacgtgcgttcaaattggaaagacgttacagagataatcgcag                - cacaattactgtatacaataaaaaaatatggtccagatcgaatcgcaggatttacaccta                - ttccagcgatgtcaatgattagttatgcagcaggtgctcgattcatcaatttgcttggtg                - gtgaaatgcttagtttttatgactggtatgcagatttaccacctgcctctccacaaattt                - ggggagagcaaacagatgtgcctgaatcaagtgactggtataacgcatcatacattatta                - tgtggggctctaatgtacctttaacacgtactccggatgcacattttatgacagaagtcc                - gctataaaggtacaaaagtcatttcagtagcaccagattacgcagaaaatgtgaaatttg                - cagataactggctagcaccgaatcctggttcagatgctgcaattgctcaagcaatgacac                - atgttattttacaagaacattatgttaatcaacctaatgaacgctttataaattacgcta                - aacaatatacagatatgccgtttcttatcatgctggatgaagatgaaaatggatataaag                - cgggtcgatttttaagagcgagtgacttaggtcaaacaacagagcaaggcgaatggaagc                - cagttattcatgatgcaatcagcgatagtttagtagtacctaatggcacaatgggtcaac                - gttgggaagaaggtaagaagtggaacttaaaactagaaacagaagatggttctaaaatta                - accctacattatcaatggcagaaggtggatacgaattagaaacaattcaattcccatact                - ttgatagtgatggagatgggatattcaatcgtccaattccaactcgacaagtcactttag                - caaatggtgacaaagtccgtattgctacaatttttgacttaatggcaagtcaatatggcg                - tgcgtcgttttgatcataaattagaatcaaaaggatacgacgatgcagaatcaaaatata                - cacctgcttggcaagaagccatttcaggcgtaaaacaaagtgttgtcattcaagtagcga                - aagaatttgcgcaaaacgctatcgatactgaagggcgttcaatgattatcatgggtgcgg                - gtattaaccattggtttaactcagatacgatttatcgttcaatcttaaacttagttatgt                - tatgtggctgtcaaggtgtgaatggtggcggttgggctcactatgtgggacaagaaaaat                - gtcgtccgattgaaggatggagtactgtcgcatttgcgaaagactggcaaggaccaccac                - gtttgcaaaatggaacaagttggttctattttgcaacagaccaatggaaatatgaagagt                - caaatgtagatcgtttaaaatctccattagctaaaacagaggatttaaagcatcaacacc                - cagctgattataatgttttagcagctagacttggttggttaccatcatatccacaattta                - ataaaaatagtttgttgtttgcagaagaagctaaagatgaaggcattgagtcgaatgagg                - caattttaaaacgagcgataaatgaagttaagtcaaaacaaacgcaatttgcgatagaag                - atccggatttgaaaaagaatcatccgaaatcactgtttatatggcgctcaaatctaatct                - caagttctgcaaaaggtcaagaatactttatgaagcatttacttggcacaaaatcagggt                - tattagctacaccaaatgaagatgaaaagccagaagaaattacgtggcgtgaggaaacaa                - cagggaaattagatttagtcgtttctttagatttcagaatgacagcaacacctttatatt                - ctgacattgttttgccagcagcgacttggtatgagaagcatgatttgtcatctacagata                - tgcatccatatgtacatccttttaatccagctattgatccattatgggaatcgcgttcag                - actgggatatttataaaacgttggcaaaagcattttcagaaatggcaaaagactatttac                - ctggaacgtttaaagatgttgtgacaactccacttagtcatgatacaaagcaagaaattt                - caacaccatacggcgtagtgaaagattggtcgaagggtgaaattgaagcggtacctggac                - gtacaatgcctaactttgcaattgtagaacgcgactacactaaaatttacgacaaatatg                - tcacgcttggtcctgtacttgaaaaagggaaagttggagcacatggtgtaagtttcggtg                - tcagtgaacaatatgaagaattaaaaagtatgttaggtacgtggagtgatacaaatgatg                - attctgtgagagcgaatcgtccgcgtattgatacagcacgtaatgtagcagatgcaatac                - taagtatttcatctgctacgaatggtaaattatcacaaaaatcatatgaagatcttgaag                - aacaaactggaatgccgttaaaagatatttctagcgaacgtgctgctgagaaaatttcgt                - ttttaaatataacttcacaaccacgagaagtaataccgacagcagtattcccaggttcaa                - ataaacaaggtcgacgatattcaccatttacaacgaatatagaacgtctagtacctttta                - gaacattaacaggacgtcaaagttattatgtggatcacgaagttttccaacaatttgggg                - agagcttaccagtatataaaccgacattgccgccaatggtatttgggaatagagataaga                - aaattaaaggtggtacagatgctttggtactgcgttatttaacgcctcatggaaaatgga                - atatacactcaatgtatcaagataataagcatatgttgacactatttagaggtggtccac                - cggtttggatatcaaatgaagatgctgaaaaacacgatatccaagataatgattggctag                - aagtgtataaccgtaatggtgttgtaacggcaagagcagttatttcgcatcgtatgccta                - aaggtacaatgtttatgtatcatgcacaagataaacatattcaaacgcctgggtcagaaa                - ttacagatacacgtggtggttcacacaacgcgccgactagaatccatttgaaaccaacac                - aactagtcggaggatacgcacaaattagttatcactttaattattatggaccaattggga                - accaaagggatttatatgtagcagttagaaagatgaaggaggttaattggcttgaagatt                - aaagcgcaagttgcgatggtattaaatttagataaatgcataggatgccatacgtgtagt                - gtgacatgtaaaaacacttggacaaatcgtccaggtgctgagtacatgtggttcaataac                - gtagaaacgaagccaggtgtagggtatccgaaacgttgggaagaccaagaacactacaaa                - ggtggttggg-3'                                                               - (B) Staphylococcus aureus respiratory nitrate reductase alpha              subunit polypeptide sequence deduced from a polynucleotide                    sequence in this table [SEQ ID NO:2].                                         NH.sub.2 -                                                                    MGKFGLNFFKPTEKFNGNWSILESKSREWEKMYRERWSHDKEVRTTHGVNCTGSCSWKVFVKNG               - VINWENQQTDYPSCGP                                                            - DMPEYEPRGCPRGASFSWYEYSPLRIKYPYIRGKLWDLWTEALEENYGNRVAAWASIVENEDKA            - KQYKQARGMGGHVRSN                                                            - WKDVTEIIAAQLLYTIKKYGPDRIAGFTPIPAMSMISYAAGARFINLLGGEMLSFYDWYADLPP            - ASPQIWGEQTDVPESS                                                            - DWYNASYIIMWGSNVPLTRTPDAHFMTEVRYKGTKVISVAPDYAENVKFADNWLAPNPGSDAAI            - AQAMTHVILQEHYVNQ                                                            - PNERFINYAKQYTDMPFLIMLDEDENGYKAGRFLRASDLGQTTEQGEWKPVIHDAISDSLVVPN            - GTMGQRWEEGKKWNLK                                                            - LETEDGSKINPTLSMAEGGYELETIQFPYFDSDGDGIFNRPIPTRQVTLANGDKVRIATIFDLM            - ASQYGVRRFDHKLESK                                                            - GYDDAESKYTPAWQEAISGVKQSVVIQVAKEFAQNAIDTEGRSMIIMGAGINHWFNSDTIYRSI            - LNLVMLCGCQGVNGGG                                                            - WAHYVGQEKCRPIEGWSTVAFAKDWQGPPRLQNGTSWFYFATDQWKYEESNVDRLKSPLAKTED            - LKHQHPADYNVLAARL                                                            - GWLPSYPQFNKNSLLFAEEAKDEGIESNEAILKRAINEVKSKQTQFAIEDPDLKKNHPKSLFIW            - RSNLISSSAKGQEYFM                                                            - KHLLGTKSGLLATPNEDEKPEEITWREETTGKLDLVVSLDFRMTATPLYSDIVLPAATWYEKHD            - LSSTDMHPYVHPFNPA                                                            - IDPLWESRSDWDIYKTLAKAFSEMAKDYLPGTFKDVVTTPLSHDTKQEISTPYGVVKDWSKGEI            - EAVPGRTMPNFAIVER                                                            - DYTKIYDKYVTLGPVLEKGKVGAHGVSFGVSEQYEELKSMLGTWSDTNDDSVRANRPRIDTARN            - VADAILSISSATNGKL                                                            - SQKSYEDLEEQTGMPLKDISSERAAEKISFLNITSQPREVIPTAVFPGSNKQGRRYSPFTTNIE            - RLVPFRTLTGRQSYYV                                                            - DHEVFQQFGESLPVYKPTLPPMVFGNRDKKIKGGTDALVLRYLTPHGKWNIHSMYQDNKHMLTL            - FRGGPPVWISNEDAEK                                                            - HDIQDNDWLEVYNRNGVVTARAVISHRMPKGTMFMYHAQDKHIQTPGSEITDTRGGSHNAPTRI            - HLKPTQLVGGYAQISY                                                            - HFNYYGPIGNQRDLYVAVRKMKEVNWLED-COOH                                          - (C) Staphylococcus aureus respiratory nitrate reductase alpha              subunit polynucleotide sequence [SEQ ID NO:3].                                5' - atgggaaaatttggattgaatttcttta                                              - agccaacagaaaaatttaatgggaattggtcgatcctagaaagtaaaagtagagaatggg                - aaaaaatgtacagagaacgttggagccacgataaagaagtaagaacaacacatggtgtta                - actgtacaggctcatgttcttggaaagtatttgtgaaaaatggtgtgattaactgggaaa                - atcaacaaactgactatccaagttgtggtccggatatgcctgaatatgaaccgagaggat                - gtccacgaggtgcgtcattctcttggtatgaatacagtccgcttcgaatcaaatatccat                - atattcgtggaaaactctgggatttatggactgaagcattagaagaaaactatggtaatc                - gcgttgctgcatgggcgtctattgttgaaaatgaagacaaagccaaacaatataagcaag                - cccgaggtatgggagggcacgtgcgttcaaattggaaagacgttacagagataatcgcag                - cacaattactgtatacaataaaaaaatatggtccagatcgaatcgcaggatttacaccta                - ttccagcgatgtcaatgattagttatgcagcaggtgctcgattcatcaatttgcttggtg                - gtgaaatgcttagtttttatgactggtatgcagatttaccacctgcctctccacaaattt                - ggggagagcaaacagatgtgcctgaatcaagtgactggtataacgcatcatacattatta                - tgtggggctctaatgtacctttaacacgtactccggatgcacattttatgacagaagtcc                - gctataaaggtacaaaagtcatttcagtagcaccagattacgcagaaaatgtgaaatttg                - cagataactggctagcaccgaatcctggttcagatgctgcaattgctcaagcaatgacac                - atgttattttacaagaacattatgttaatcaacctaatgaacgctttataaattacgcta                - aacaatatacagatatgccgtttcttatcatgctggatgaagatgaaaatggatataaag                - cgggtcgatttttaagagcgagtgacttaggtcaaacaacagagcaaggcgaatggaagc                - cagttattcatgatgcaatcagcgatagtttagtagtacctaatggcacaatgggtcaac                - gttgggaagaaggtaagaagtggaacttaaaactagaaacagaagatggttctaaaatta                - accctacattatcaatggcagaaggtggatacgaattagaaacaattcaattcccatact                - ttgatagtgatggagatgggatattcaatcgtccaattccaactcgacaagtcactttag                - caaatggtgacaaagtccgtattgctacaatttttgacttaatggcaagtcaatatggcg                - tgcgtcgttttgatcataaattagaatcaaaaggatacgacgatgcagaatcaaaatata                - cacctgcttggcaagaagccatttcaggcgtaaaacaaagtgttgtcattcaagtagcga                - aagaatttgcgcaaaacgctatcgatactgaagggcgttcaatgattatcatgggtgcgg                - gtattaaccattggtttaactcagatacgatttatcgttcaatcttaaacttagttatgt                - tatgtggctgtcaaggtgtgaatggtggcggttgggctcactatgtgggacaagaaaaat                - gtcgtccgattgaaggatggagtactgtcgcatttgcgaaagactggcaaggaccaccac                - gtttgcaaaatggaacaagttggttctattttgcaacagaccaatggaaatatgaagagt                - caaatgtagatcgtttaaaatctccattagctaaaacagaggatttaaagcatcaacacc                - cagctgattataatgttttagcagctagacttggttggttaccatcatatccacaattta                - ataaaaatagtttgttgtttgcagaagaagctaaagatgaaggcattgagtcgaatgagg                - caattttaaaacgagcgataaatgaagttaagtcaaaacaaacgcaatttgcgatagaag                - atccggatttgaaaaagaatcatccgaaatcactgtttatatggcgctcaaatctaatct                - caagttctgcaaaaggtcaagaatactttatgaagcatttacttggcacaaaatcagggt                - tattagctacaccaaatgaagatgaaaagccagaagaaattacgtggcgtgaggaaacaa                - cagggaaattagatttagtcgtttctttagatttcagaatgacagcaacacctttatatt                - ctgacattgttttgccagcagcgacttggtatgagaagcatgatttgtcatctacagata                - tgcatccatatgtacatccttttaatccagctattgatccattatgggaatcgcgttcag                - actgggatatttataaaacgttggcaaaagcattttcagaaatggcaaaagactatttac                - ctggaacgtttaaagatgttgtgacaactccacttagtcatgatacaaagcaagaaattt                - caacaccatacggcgtagtgaaagattggtcgaagggtgaaattgaagcggtacctggac                - gtacaatgcctaactttgcaattgtagaacgcgactacactaaaatttacgacaaatatg                - tcacgcttggtcctgtacttgaaaaagggaaagttggagcacatggtgtaagtttcggtg                - tcagtgaacaatatgaagaattaaaaagtatgttaggtacgtggagtgatacaaatgatg                - attctgtgagagcgaatcgtccgcgtattgatacagcacgtaatgtagcagatgcaatac                - taagtatttcatctgctacgaatggtaaattatcacaaaaatcatatgaagatcttgaag                - aacaaactggaatgccgttaaaagatatttctagcgaacgtgctgctgagaaaatttcgt                - ttttaaatataacttcacaaccacgagaagtaataccgacagcagtattcccaggttcaa                - ataaacaaggtcgacgatattcaccatttacaacgaatatagaacgtctagtacctttta                - gaacattaacaggacgtcaaagttattatgtggatcacgaagttttccaacaatttgggg                - agagcttaccagtatataaaccgacattgccgccaatggtatttgggaatagagataaga                - aaattaaaggtggtacagatgctttggtactgcgttatttaacgcctcatggaaaatgga                - atatacactcaatgtatcaagataataagcatatgttgacactatttagaggtggtccac                - cggtttggatatcaaatgaagatgctgaaaaacacgatatccaagataatgattggctag                - aagtgtataaccgtaatggtgttgtaacggcaagagcagttatttcgcatcgtatgccta                - aaggtacaatgtttatgtatcatgcacaagataaacatattcaaacgcctgggtcagaaa                - ttacagatacacgtggtggttcacacaacgcgccgactagaatccatttgaaaccaacac                - aactagtcggaggatacgcacaaattagttatcactttaattattatggaccaattggga                - accaaagggatttatatgtagcagttagaaagatgaaggaggttaattggcttgaagatt                - aa-3'                                                                     __________________________________________________________________________

Deposited materials

A deposit comprising a Staphylococcus aureus WCUH 29 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 1 RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771, andreferred to as Staphylococcus aureus WCUH29 on deposit. TheStaphylococcus aureus strain deposit is referred to herein as "thedeposited strain" or as "the DNA of the deposited strain."

The deposited strain comprises a full length respiratory nitratereductase alpha subunit gene. The sequence of the polynucleotidescomprised in the deposited strain, as well as the amino acid sequence ofany polypeptide encoded thereby, are controlling in the event of anyconflict with any description of sequences herein.

The deposit of the deposited strain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The deposited strainwill be irrevocably and without restriction or condition released to thepublic upon the issuance of a patent. The deposited strain is providedmerely as convenience to those of skill in the art and is not anadmission that a deposit is required for enablement, such as thatrequired under 35 U.S.C. §112. A license may be required to make, use orsell the deposited strain, and compounds derived therefrom, and no suchlicense is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStaphylococcus aureus WCUH 29 strain, which polypeptide is comprised inthe deposited strain. Further provided by the invention are respiratorynitrate reductase alpha subunit polynucleotide sequences in thedeposited strain, such as DNA and RNA, and amino acid sequences encodedthereby. Also provided by the invention are respiratory nitratereductase alpha subunit polypeptide and polynucleotide sequencesisolated from the deposited strain.

Polypeptides

Respiratory nitrate reductase alpha subunit polypeptide of the inventionis substantially phylogenetically related to other proteins of thereductases family.

In one aspect of the invention there are provided polypeptides ofStaphylococcus aureus referred to herein as "respiratory nitratereductase alpha subunit" and "respiratory nitrate reductase alphasubunit polypeptides" as well as biologically, diagnostically,prophylactically, clinically or therapeutically useful variants thereof,and compositions comprising the same.

Among the particularly preferred embodiments of the invention arevariants of respiratory nitrate reductase alpha subunit polypeptideencoded by naturally occurring alleles of a respiratory nitratereductase alpha subunit gene.

The present invention further provides for an isolated polypeptide that:(a) comprises or consists of an amino acid sequence that has at least95% identity, most preferably at least 97-99% or exact identity, to thatof SEQ ID NO:2 over the entire length of SEQ ID NO:2; (b) a polypeptideencoded by an isolated polynucleotide comprising or consisting of apolynucleotide sequence that has at least 95% identity, even morepreferably at least 97-99% or exact identity to SEQ ID NO:1 over theentire length of SEQ ID NO:1, or the entire length of that portion ofSEQ ID NO:1 which encodes SEQ ID NO:2; (c) a polypeptide encoded by anisolated polynucleotide comprising or consisting of a polynucleotidesequence encoding a polypeptide that has at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2] (in particular a mature polypeptide) as well as polypeptidesand fragments, particularly those that has a biological activity ofrespiratory nitrate reductase alpha subunit, and also those that have atleast 95% identity to a polypeptide of Table 1 [SEQ ID NO:2] and alsoinclude portions of such polypeptides with such portion of thepolypeptide generally comprising at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

    X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStaphylococcus aureus, however, it may preferably be obtained from otherorganisms of the same taxonomic genus. A polypeptide of the inventionmay also be obtained, for example, from organisms of the same taxonomicfamily or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with respiratory nitrate reductasealpha subunit polypeptides, fragments may be "free-standing," orcomprised within a larger polypeptide of which they form a part orregion, most preferably as a single continuous region in a single largerpolypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Staphylococcus aureus, are also preferred. Furtherpreferred are fragments characterized by structural or functionalattributes such as fragments that comprise alpha-helix and alpha-helixforming regions, beta-sheet and beta-sheet-forming regions, turn andturn-forming regions, coil and coil-forming regions, hydrophilicregions, hydrophobic regions, alpha amphipathic regions, betaamphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO:2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

Polynucleotides

It is an object of the invention to provide polynucleotides that encoderespiratory nitrate reductase alpha subunit polypeptides, particularlypolynucleotides that encode a polypeptide herein designated respiratorynitrate reductase alpha subunit.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding respiratory nitrate reductasealpha subunit polypeptides comprising a sequence set out in Table 1 [SEQID NO:1, 3] that includes a full length gene, or a variant thereof. TheApplicants believe that this full length gene is essential to the growthand/or survival of an organism that possesses it, such as Staphylococcusaureus.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing respiratory nitrate reductasealpha subunit polypeptides and polynucleotides, particularlyStaphylococcus aureus respiratory nitrate reductase alpha subunitpolypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

Another aspect of the invention relates to isolated polynucleotides,including at least one full length gene, that encodes a respiratorynitrate reductase alpha subunit polypeptide having a deduced amino acidsequence of Table 1 [SEQ ID NO:2] and polynucleotides closely relatedthereto and variants thereof.

In another particularly preferred embodiment of the invention there is arespiratory nitrate reductase alpha subunit polypeptide fromStaphylococcus aureus comprising or consisting of an amino acid sequenceof Table 1 [SEQ ID NO:2], or a variant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO:1, 3], a polynucleotide of the inventionencoding respiratory nitrate reductase alpha subunit polypeptide may beobtained using standard cloning and screening methods, such as those forcloning and sequencing chromosomal DNA fragments from bacteria usingStaphylococcus aureus WCUH 29 cells as starting material, followed byobtaining a full length clone. For example, to obtain a polynucleotidesequence of the invention, such as a polynucleotide sequence given inTable 1 [SEQ ID NO:1, 3], typically a library of clones of chromosomalDNA of Staphylococcus aureus WCUH 29 in E. coli or some other suitablehost is probed with a radiolabeled oligonucleotide, preferably a 17-meror longer, derived from a partial sequence. Clones carrying DNAidentical to that of the probe can then be distinguished using stringenthybridization conditions. By sequencing the individual clones thusidentified by hybridization with sequencing primers designed from theoriginal polypeptide or polynucleotide sequence it is then possible toextend the polynucleotide sequence in both directions to determine afull length gene sequence. Conveniently, such sequencing is performed,for example, using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York(1989). (see in particular Screening By Hybridization 1.90 andSequencing Denatured Double-Stranded DNA Templates 13.70). Directgenomic DNA sequencing may also be performed to obtain a full lengthgene sequence. Illustrative of the invention, each polynucleotide setout in Table 1 [SEQ ID NO:1, 3] was discovered in a DNA library derivedfrom Staphylococcus aureus WCUH 29.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO:1, 3] containsan open reading frame encoding a protein having about the number ofamino acid residues set forth in Table 1 [SEQ ID NO:2] with a deducedmolecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art. Thepolynucleotide of SEQ ID NO:1, between nucleotide number 453 (ATG) andthe codon that begins at nucleotide number 4142 (TAA) of SEQ ID NO:1,encodes the polypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of: (a) a polynucleotidesequence that has at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ IDNO:1, or the entire length of that portion of SEQ ID NO:1 which encodesSEQ ID NO:2; (b) a polynucleotide sequence encoding a polypeptide thathas at least 95% identity, even more preferably at least 97-99% or 100%exact, to the amino acid sequence of SEQ ID NO:2, over the entire lengthof SEQ ID NO:2.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Staphylococcusaureus, may be obtained by a process that comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO:1, 3 or a fragment thereof; andisolating a full-length gene and/or genomic clones comprising saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1, 3]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also comprise at least onenon-coding sequence, including for example, but not limited to at leastone non-coding 5' and 3' sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of a fused polypeptide can be encoded. Incertain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof that may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 459 (ATG) to the nucleotideimmediately upstream of or including nucleotide 4146 (TAA) set forth inSEQ ID NO:1, 3 of Table 1, both of that encode a respiratory nitratereductase alpha subunit polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

    X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y

wherein, at the 5' end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3' end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero , n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5' end nucleic acid residue is at the left,bound to R₁, and its 3' end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, that can be adouble-stranded polynucleotide wherein the formula shows a first strandto which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Staphylococcus aureus, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polynucleotide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Staphylococcus aureus respiratorynitrate reductase alpha subunit having an amino acid sequence set out inTable 1 [SEQ ID NO:2]. The term also encompasses polynucleotides thatinclude a single continuous region or discontinuous regions encoding thepolypeptide (for example, polynucleotides interrupted by integratedphage, an integrated insertion sequence, an integrated vector sequence,an integrated transposon sequence, or due to RNA editing or genomic DNAreorganization) together with additional regions, that also may comprisecoding and/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2]. Fragments ofpolynucleotides of the invention may be used, for example, tosyrffiesize full-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingrespiratory nitrate reductase alpha subunit variants, that have theamino acid sequence of respiratory nitrate reductase alpha subunitpolypeptide of Table 1 [SEQ ID NO:2] in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified,deleted and/or added, in any combination. Especially preferred amongthese are silent substitutions, additions and deletions, that do notalter the properties and activities of respiratory nitrate reductasealpha subunit polypeptide.

Preferred isolated polynucleotide embodiments also includepolynucleotide fragments, such as a polynucleotide comprising a nuclicacid sequence having at least 15, 20, 30, 40, 50 or 100 contiguousnucleic acids from the polynucleotide sequence of SEQ ID NO:1, 3, or anpolynucleotide comprising a nucleic acid sequence having at least 15,20, 30, 40, 50 or 100 contiguous nucleic acids truncated or deleted fromthe 5' and/or 3' end of the polynucleotide sequence of SEQ ID NO:1, 3.

Further preferred embodiments of the invention are polynucleotides thatare at least 95% or 97% identical over their entire length to apolynucleotide encoding respiratory nitrate reductase alpha subunitpolypeptide having an amino acid sequence set out in Table 1 [SEQ IDNO:2], and polynucleotides that are complementary to suchpolynucleotides. Most highly preferred are polynucleotides that comprisea region that is at least 95% are especially preferred. Furthermore,those with at least 97% are highly preferred among those with at least95%, and among these those with at least 98% and at least 99% areparticularly highly preferred, with at least 99% being the morepreferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as amature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1,3].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to respiratory nitrate reductase alpha subunitpolynucleotide sequences, such as those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms "stringent conditions" and "stringent hybridization conditions"mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1× SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycomprising a complete gene for a polynucleotide sequence set forth inSEQ ID NO:1, 3 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO:1, 3 or a fragment thereof, and isolating said polynucleotidesequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefill-length cDNAs and genomic clones encoding respiratory nitratereductase alpha subunit and to isolate cDNA and genomic clones of othergenes that have a high identity, particularly high sequence identity, toa respiratory nitrate reductase alpha subunit gene. Such probesgenerally will comprise at least 15 nucleotide residues or base pairs.Preferably, such probes will have at least 30 nucleotide residues orbase pairs and may have at least 50 nucleotide residues or base pairs.Particularly preferred probes will have at least 20 nucleotide residuesor base pairs and will have lee than 30 nucleotide residues or basepairs.

A coding region of a respiratory nitrate reductase alpha subunit genemay be isolated by screening using a DNA sequence provided in Table 1[SEQ ID NO:1, 3] to synthesize an oligonucleotide probe. A labeledoligonucleotide having a sequence complementary to that of a gene of theinvention is then used to screen a library of cDNA, genomic DNA or mRNAto determine which members of the library the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an`adaptor` sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the "missing" 5' end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using"nested" primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3' in the adaptor sequence and a gene specific primer thatanneals further 5' in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5' primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2] may be used in theprocesses herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is a mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to a mature polypeptide (when amature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from a mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

As will be recognized, the entire polypeptide encoded by an open readingframe is often not required for activity. Accordingly, it has becomeroutine in molecular biology to map the boundaries of the primarystructure required for activity with N-terminal and C-terminal deletionexperiments. These experiments utilize exonuclease digestion orconvenient restriction sites to cleave coding nucleic acid sequence. Forexample, Promega (Madison, Wis.) sell an Erase-a-base™ system that usesExonuclease III designed to facilitate analysis of the deletion products(protocol available at www.promega.com). The digested endpoints can berepaired (e.g., by ligation to synthetic linkers) to the extentnecessary to preserve an open reading frame. In this way, the nucleicacid of SEQ ID NO:1, 3 readily provides contiguous fragments of SEQ IDNO:2 sufficient to provide an activity, such as an enzymatic, binding orantibody-inducing activity. Nucleic acid sequences encoding suchfragments of SEQ ID NO:2 and variants thereof as described herein arewithin the invention, as are polypeptides so encoded.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, that is a precursor to a proprotein, having a leadersequence and one or more prosequences, that generally are removed duringprocessing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells that are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci E. coli,streptomyces, cyanobacteria, Bacillus subtilis, and Staphylococcusaureus; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may comprise control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of respiratory nitratereductase alpha subunit polynucleotides and polypeptides of theinvention for use as diagnostic reagents. Detection of respiratorynitrate reductase alpha subunit polynucleotides and/or polypeptides in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of disease, staging of disease orresponse of an infectious organism to drugs. Eukaryotes, particularlymammals, and especially humans, particularly those infected or suspectedto be infected with an organism comprising the respiratory nitratereductase alpha subunit gene or protein, may be detected at the nucleicacid or amino acid level by a variety of well known techniques as wellas by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genomicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled respiratory nitrate reductasealpha subunit polynucleotide sequences. Perfectly or significantlymatched sequences can be distinguished from imperfectly or moresignificantly mismatched duplexes by DNase or RNase digestion, for DNAor RNA respectively, or by detecting differences in melting temperaturesor renaturation kinetics. Polynucleotide sequence differences may alsobe detected by alterations in the electrophoretic mobility ofpolynucleotide fragments in gels as compared to a reference sequence.This may be carried out with or without denaturing agents.Polynucleotide differences may also be detected by direct DNA or RNAsequencing. See, for example, Myers et al., Science, 230: 1242 (1985).Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase, V1 and S1 protection assay or achemical cleavage method. See, for example, Cotton et al., Proc. Natl.Acad. Sci., USA, 85: 4397-4401(1985).

In another embodiment, an array of oligonucleotides probes comprisingrespiratory nitrate reductase alpha subunit nucleotide sequence orfragments thereof can be constructed to conduct efficient screening of,for example, genetic mutations, serotype, taxonomic classification oridentification. Array technology methods are well known and have generalapplicability and can be used to address a variety of questions inmolecular genetics including gene expression, genetic linkage, andgenetic variability (see, for example, Chee et al., Science, 274: 610(1996)).

Thus in another aspect, the present invention relates to a diagnostickit that comprises: (a) a polynucleotide of the present invention,preferably the nucleotide sequence of SEQ ID NO:1, 3, or a fragmentthereof; (b) a nucleotide sequence complementary to that of (a); (c) apolypeptide of the present invention, preferably the polypeptide of SEQID NO:2 or a fragment thereof; or (d) an antibody to a polypeptide ofthe present invention, preferably to the polypeptide of SEQ ID NO:2. Itwill be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1, 3, that isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, that results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding respiratory nitrate reductase alpha subunitpolypeptide can be used to identify and analyze mutations. The inventionfurther provides these primers with 1, 2, 3 or 4 nucleotides removedfrom the 5' and/or the 3' end. These primers may be used for, amongother things, amplifying respiratory nitrate reductase alpha subunit DNAand/or RNA isolated from a sample derived from an individual, such as abodily material. The primers may be used to amplify a polynucleotideisolated from an infected individual, such that the polynucleotide maythen be subject to various techniques for elucidation of thepolynucleotide sequence. In this way, mutations in the polynucleotidesequence may be detected and used to diagnose and/or prognose theinfection or its stage or course, or to serotype and/or classify theinfectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStaphylococcus aureus, comprising determining from a sample derived froman individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO:1,3]. Increased or decreased expression of a respiratory nitrate reductasealpha subunit polynucleotide can be measured using any on of the methodswell known in the art for the quantitation of polynucleotides, such as,for example, amplification, PCR, RT-PCR, RNase protection, Northernblotting, spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of respiratory nitrate reductase alpha subunitpolypeptide compared to normal control tissue samples may be used todetect the presence of an infection, for example. Assay techniques thatcan be used to determine levels of a respiratory nitrate reductase alphasubunit polypeptide, in a sample derived from a host, such as a bodilymaterial, are well-known to those of skill in the art. Such assaymethods include radioimmunoassays, competitive-binding assays, WesternBlot analysis, antibody sandwich assays, antibody detection and ELISAassays.

Antagonists and Agonists--Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases herein mentioned. It is thereforedesirable to devise screening methods to identify compounds that agonize(e.g., stimulate) or that antagonize (e.g.,inhibit) the function of thepolypeptide or polynucleotide. Accordingly, in a further aspect, thepresent invention provides for a method of screening compounds toidentify those that agonize or that antagonize the function of apolypeptide or polynucleotide of the invention, as well as relatedpolypeptides and polynucleotides. In general, agonists or antagonists(e.g., inhibitors) may be employed for therapeutic and prophylacticpurposes for such Diseases as herein mentioned. Compounds may beidentified from a variety of sources, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Suchagonists and antagonists so-identified may be natural or modifiedsubstrates, ligands, receptors, enzymes, etc., as the case may be, ofrespiratory nitrate reductase alpha subunit polypeptides andpolynucleotides; or may be structural or functional mimetics thereof(see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists, in the absence of an agonist or antagonist, by testing whetherthe candidate compound results in inhibition of activation of thepolypeptide or polynucleotide, as the case may be. Further, thescreening methods may simply comprise the steps of mixing a candidatecompound with a solution comprising a polypeptide or polynucleotide ofthe present invention, to form a mixture, measuring respiratory nitratereductase alpha subunit polypeptide and/or polynucleotide activity inthe mixture, and comparing the respiratory nitrate reductase alphasubunit polypeptide and/or polynucleotide activity of the mixture to astandard. Fusion proteins, such as those made from Fc portion andrespiratory nitrate reductase alpha subunit polypeptide, as hereindescribed, can also be used for high-throughput screening assays toidentify antagonists of the polypeptide of the present invention, aswell as of phylogenetically and and/or functionally related polypeptides(see D. Bennett et al., J Mol Recognition, 8:52-58 (1995); and K.Johanson et al., J Biol Chem, 270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentsthat may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose that enhance (agonist) or block (antagonist) the action ofrespiratory nitrate reductase alpha subunit polypeptides orpolynucleotides, particularly those compounds that are bacteristaticand/or bactericidal. The method of screening may involve high-throughputtechniques. For example, to screen for agonists or antagonists, asynthetic reaction mix, a cellular compartment, such as a membrane, cellenvelope or cell wall, or a preparation of any thereof, comprisingrespiratory nitrate reductase alpha subunit polypeptide and a labeledsubstrate or ligand of such polypeptide is incubated in the absence orthe presence of a candidate molecule that may be a respiratory nitratereductase alpha subunit agonist or antagonist. The ability of thecandidate molecule to agonize or antagonize the respiratory nitratereductase alpha subunit polypeptide is reflected in decreased binding ofthe labeled ligand or decreased production of product from suchsubstrate. Molecules that bind gratuitously, i.e., without inducing theeffects of respiratory nitrate reductase alpha subunit polypeptide aremost likely to be good antagonists. Molecules that bind well and, as thecase may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in respiratory nitrate reductase alphasubunit polynucleotide or polypeptide activity, and binding assays knownin the art.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance, ¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide that compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by respiratory nitrate reductase alpha subunitpolypeptide associating with another respiratory nitrate reductase alphasubunit polypeptide or other polypeptide, labeled to comprise afluorescently-labeled molecule will have higher polarization values thana fluorescently labeled monomeric protein. It is preferred that thismethod be used to characterize small molecules that disrupt polypeptidecomplexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of respiratory nitratereductase alpha subunit polypeptide dimers, trimers, tetramers or higherorder structures, or structures formed by respiratory nitrate reductasealpha subunit polypeptide bound to another polypeptide. Respiratorynitrate reductase alpha subunit polypeptide can be labeled with both adonor and acceptor fluorophore. Upon mixing of the two labeled speciesand excitation of the donor fluorophore, fluorescence energy transfercan be detected by observing fluorescence of the acceptor. Compoundsthat block dimerization will inhibit fluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on respiratory nitrate reductase alpha subunit polypeptideself-association as well as an association of respiratory nitratereductase alpha subunit polypeptide and another polypeptide or smallmolecule. Respiratory nitrate reductase alpha subunit polypeptide can becoupled to a sensor chip at low site density such that covalently boundmolecules will be monomeric. Solution protein can then passed over therespiratory nitrate reductase alpha subunit polypeptide -coated surfaceand specific binding can be detected in real-time by monitoring thechange in resonance angle caused by a change in local refractive index.This technique can be used to characterize the effect of small moleculeson kinetic rates and equilibrium binding constants for respiratorynitrate reductase alpha subunit polypeptide self-association as well asan association of respiratory nitrate reductase alpha subunitpolypeptide and another polypeptide or small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of respiratory nitrate reductasealpha subunit polypeptide with another respiratory nitrate reductasealpha subunit polypeptide or a different polypeptide. Respiratorynitrate reductase alpha subunit polypeptide can be coupled to ascintillation-filled bead. Addition of radio-labeled respiratory nitratereductase alpha subunit polypeptide results in binding where theradioactive source molecule is in close proximity to the scintillationfluid. Thus, signal is emitted upon respiratory nitrate reductase alphasubunit polypeptide binding and compounds that prevent respiratorynitrate reductase alpha subunit polypeptide self-association or anassociation of respiratory nitrate reductase alpha subunit polypeptideand another polypeptide or small molecule will diminish signal.

In other embodiments of the invention there are provided methods foridentify compounds that bind to or otherwise interact with and inhibitor activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for respiratory nitrate reductase alphasubunit agonists is a competitive assay that combines respiratorynitrate reductase alpha subunit and a potential agonist with respiratorynitrate reductase alpha subunit-binding molecules, recombinantrespiratory nitrate reductase alpha subunit binding molecules, naturalsubstrates or ligands, or substrate or ligand mimetics, underappropriate conditions for a competitive inhibition assay. Respiratorynitrate reductase alpha subunit can be labeled, such as by radioactivityor a calorimetric compound, such that the number of respiratory nitratereductase alpha subunit molecules bound to a binding molecule orconverted to product can be determined accurately to assess theeffectiveness of the potential antagonist.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist or antagonist of thepolypeptide and/or polynucleotide, by: (a) determining in the firstinstance the three-dimensional structure of the polypeptide and/orpolynucleotide, or complexes thereof; (b) deducing the three-dimensionalstructure for the likely reactive site(s), binding site(s) or motif(s)of an agonist or antagonist; (c) synthesizing candidate compounds thatare predicted to bind to or react with the deduced binding site(s),reactive site(s), and/or motif(s); and (d) testing whether the candidatecompounds are indeed agonists or antagonists.

It will be further appreciated that this will normally be an iterativeprocess, and this iterative process may be performed using automated andcomputer-controlled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of respiratory nitrate reductase alpha subunitpolypeptide and/or polynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of therespiratory nitrate reductase alpha subunit polypeptide and/orpolypeptide.

In still another approach, expression of the gene encoding endogenousrespiratory nitrate reductase alpha subunit polypeptide can be inhibitedusing expression blocking techniques. This blocking may be targetedagainst any step in gene expression, but is preferably targeted againsttranscription and/or translation. An examples of a known technique ofthis sort involve the use of antisense sequences, either internallygenerated or separately administered (see, for example, O'Connor, JNeurochem (1991) 56:560 in Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Alternatively,oligonucleotides that form triple helices with the gene can be supplied(see, for example, Lee et al., Nucleic Acids Res (1979) 6:3073; Cooneyet al., Science (1988) 241:456; Dervan et al., Science (1991) 251:1360).These oligomers can be administered per se or the relevant oligomers canbe expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial respiratory nitrate reductase alpha subunit proteins thatmediate tissue damage and/or; to block the normal progression ofpathogenesis in infections initiated other than by the implantation ofin-dwelling devices or by other surgical techniques.

In accordance with yet another aspect of the invention, there areprovided respiratory nitrate reductase alpha subunit agonists andantagonists, preferably bacteristatic or bactericidal agonists andantagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer (1994)Schistosomes, Liver Flukes and Helicobacter pylori (International Agencyfor Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofrespiratory nitrate reductase alpha subunit polypeptides and/orpolynucleotides) found using screens provided by the invention, or knownin the art, particularly narrow-spectrum antibiotics, should be usefulin the treatment of H. pylori infection. Such treatment should decreasethe advent of H. pylori-induced cancers, such as gastrointestinalcarcinoma. Such treatment should also prevent, inhibit and/or curegastric ulcers and gastritis.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

GLOSSARY

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

"Disease(s)" means any disease caused by or related to infection by abacteria, including, for example, disease, such as, infections of theupper respiratory tract (e.g., otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g., empyema, lungabscess), cardiac (e.g., infective endocarditis), gastrointestinal(e.g., secretory diarrhoea, splenic absces, retroperitoneal abscess),CNS (e.g., cerebral abscess), eye (e.g., blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g., epididymitis,intrarenal and perinephric absces, toxic shock syndrome), skin (e.g.,impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g., septic arthrtis,osteomyelitis).

"Host cell(s)" is a cell that has been introduced (e.g., transformed ortransfected) or is capable of introduction (e.g., transformation ortransfection) by an exogenous polynucleotide sequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,"identity" also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. "Identity"can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a95, 97 or 100% identity to the reference sequence of SEQ ID NO: 1,wherein said polynucleotide sequence may be identical to the referencesequence of SEQ ID NO: 1 or may include up to a certain integer numberof nucleotide alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least onenucleotide deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5' or 3' terminal positions of the reference nucleotide sequence oranywhere between those terminal positions, interspersed eitherindividually among the nucleotides in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of nucleotide alterations is determined by multiplying thetotal number of nucleotides in SEQ ID NO:1 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of nucleotides in SEQ ID NO:1 or:

    n.sub.n ≦x.sub.n -(x.sub.n ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO: 1, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(n) and y is roundeddown to the nearest integer prior to subtracting it from x_(n).Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO:2 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations.

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 95, 97 or 100% identity to apolypeptide reference sequence of SEQ ID NO:2, wherein said polypeptidesequence may be identical to the reference sequence of SEQ ID NO:2 ormay include up to a certain integer number of amino acid alterations ascompared to the reference sequence, wherein said alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence, and whereinsaid number of amino acid alterations is determined by multiplying thetotal number of amino acids in SEQ ID NO:2 by the integer defining thepercent identity divided by 100 and then subtracting that product fromsaid total number of amino acids in SEQ ID NO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.95 for 95%, 0.97 for97% or 1.00 for 100%, and · is the symbol for the multiplicationoperator, and wherein any non-integer product of x_(a) and y is roundeddown to the nearest integer prior to subtracting it from x_(a).

"Individual(s)" means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is "isolated" even if it is still present in saidorganism, which organism may be living or non-living.

"Organism(s)" means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxyribonucleotide, that may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that comprise one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of fiavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclation, disulfide bond formation, dernethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gauma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS--STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFYCATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Recombinant expression system(s)" refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and ile; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, that arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO:1]was obtained from a library of clones of chromosomal DNA ofStaphylococcus aureus in E. coli. The sequencing data from two or moreclones comprising overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 2                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 4330                                                            <212> TYPE: DNA                                                               <213> ORGANISM: Staphylococcus aureus                                          - - <400> SEQUENCE: 1                                                         - - ctgcaggaat tcccaaatga tacatcgaat ggcacatatc attcgtcgca at -             #atgattac     60                                                                 - - tatgatgcat ttattaagca gcaagaaaat gtaacatata tttcaaccga tc -            #gtgcagat    120                                                                 - - gctaatacag tgttatgtca ctaatttata aaaaataaat gaataagtaa gg -            #tttcaacc    180                                                                 - - gagagaatat attcgtgttg aagccttatt tgtcgttgtg ccaaatttga ac -            #gatttaga    240                                                                 - - ttggcaataa gcacgaccat acatgattgt gtattatttc aatgaaatcc cg -            #ttattgat    300                                                                 - - agggaaattc cttaagtaat taggtgattc cctaatattt tatatttgtg aa -            #aaaatgta    360                                                                 - - caatctaatt aaagcaatag tcttgggcat tttaaagtat ttaaaaacac ga -            #catctaca    420                                                                 - - catcgtattt ttatacgtag gaggatataa atatgggaaa atttggattg aa -            #tttcttta    480                                                                 - - agccaacaga aaaatttaat gggaattggt cgatcctaga aagtaaaagt ag -            #agaatggg    540                                                                 - - aaaaaatgta cagagaacgt tggagccacg ataaagaagt aagaacaaca ca -            #tggtgtta    600                                                                 - - actgtacagg ctcatgttct tggaaagtat ttgtgaaaaa tggtgtgatt aa -            #ctgggaaa    660                                                                 - - atcaacaaac tgactatcca agttgtggtc cggatatgcc tgaatatgaa cc -            #gagaggat    720                                                                 - - gtccacgagg tgcgtcattc tcttggtatg aatacagtcc gcttcgaatc aa -            #atatccat    780                                                                 - - atattcgtgg aaaactctgg gatttatgga ctgaagcatt agaagaaaac ta -            #tggtaatc    840                                                                 - - gcgttgctgc atgggcgtct attgttgaaa atgaagacaa agccaaacaa ta -            #taagcaag    900                                                                 - - cccgaggtat gggagggcac gtgcgttcaa attggaaaga cgttacagag at -            #aatcgcag    960                                                                 - - cacaattact gtatacaata aaaaaatatg gtccagatcg aatcgcagga tt -            #tacaccta   1020                                                                 - - ttccagcgat gtcaatgatt agttatgcag caggtgctcg attcatcaat tt -            #gcttggtg   1080                                                                 - - gtgaaatgct tagtttttat gactggtatg cagatttacc acctgcctct cc -            #acaaattt   1140                                                                 - - ggggagagca aacagatgtg cctgaatcaa gtgactggta taacgcatca ta -            #cattatta   1200                                                                 - - tgtggggctc taatgtacct ttaacacgta ctccggatgc acattttatg ac -            #agaagtcc   1260                                                                 - - gctataaagg tacaaaagtc atttcagtag caccagatta cgcagaaaat gt -            #gaaatttg   1320                                                                 - - cagataactg gctagcaccg aatcctggtt cagatgctgc aattgctcaa gc -            #aatgacac   1380                                                                 - - atgttatttt acaagaacat tatgttaatc aacctaatga acgctttata aa -            #ttacgcta   1440                                                                 - - aacaatatac agatatgccg tttcttatca tgctggatga agatgaaaat gg -            #atataaag   1500                                                                 - - cgggtcgatt tttaagagcg agtgacttag gtcaaacaac agagcaaggc ga -            #atggaagc   1560                                                                 - - cagttattca tgatgcaatc agcgatagtt tagtagtacc taatggcaca at -            #gggtcaac   1620                                                                 - - gttgggaaga aggtaagaag tggaacttaa aactagaaac agaagatggt tc -            #taaaatta   1680                                                                 - - accctacatt atcaatggca gaaggtggat acgaattaga aacaattcaa tt -            #cccatact   1740                                                                 - - ttgatagtga tggagatggg atattcaatc gtccaattcc aactcgacaa gt -            #cactttag   1800                                                                 - - caaatggtga caaagtccgt attgctacaa tttttgactt aatggcaagt ca -            #atatggcg   1860                                                                 - - tgcgtcgttt tgatcataaa ttagaatcaa aaggatacga cgatgcagaa tc -            #aaaatata   1920                                                                 - - cacctgcttg gcaagaagcc atttcaggcg taaaacaaag tgttgtcatt ca -            #agtagcga   1980                                                                 - - aagaatttgc gcaaaacgct atcgatactg aagggcgttc aatgattatc at -            #gggtgcgg   2040                                                                 - - gtattaacca ttggtttaac tcagatacga tttatcgttc aatcttaaac tt -            #agttatgt   2100                                                                 - - tatgtggctg tcaaggtgtg aatggtggcg gttgggctca ctatgtggga ca -            #agaaaaat   2160                                                                 - - gtcgtccgat tgaaggatgg agtactgtcg catttgcgaa agactggcaa gg -            #accaccac   2220                                                                 - - gtttgcaaaa tggaacaagt tggttctatt ttgcaacaga ccaatggaaa ta -            #tgaagagt   2280                                                                 - - caaatgtaga tcgtttaaaa tctccattag ctaaaacaga ggatttaaag ca -            #tcaacacc   2340                                                                 - - cagctgatta taatgtttta gcagctagac ttggttggtt accatcatat cc -            #acaattta   2400                                                                 - - ataaaaatag tttgttgttt gcagaagaag ctaaagatga aggcattgag tc -            #gaatgagg   2460                                                                 - - caattttaaa acgagcgata aatgaagtta agtcaaaaca aacgcaattt gc -            #gatagaag   2520                                                                 - - atccggattt gaaaaagaat catccgaaat cactgtttat atggcgctca aa -            #tctaatct   2580                                                                 - - caagttctgc aaaaggtcaa gaatacttta tgaagcattt acttggcaca aa -            #atcagggt   2640                                                                 - - tattagctac accaaatgaa gatgaaaagc cagaagaaat tacgtggcgt ga -            #ggaaacaa   2700                                                                 - - cagggaaatt agatttagtc gtttctttag atttcagaat gacagcaaca cc -            #tttatatt   2760                                                                 - - ctgacattgt tttgccagca gcgacttggt atgagaagca tgatttgtca tc -            #tacagata   2820                                                                 - - tgcatccata tgtacatcct tttaatccag ctattgatcc attatgggaa tc -            #gcgttcag   2880                                                                 - - actgggatat ttataaaacg ttggcaaaag cattttcaga aatggcaaaa ga -            #ctatttac   2940                                                                 - - ctggaacgtt taaagatgtt gtgacaactc cacttagtca tgatacaaag ca -            #agaaattt   3000                                                                 - - caacaccata cggcgtagtg aaagattggt cgaagggtga aattgaagcg gt -            #acctggac   3060                                                                 - - gtacaatgcc taactttgca attgtagaac gcgactacac taaaatttac ga -            #caaatatg   3120                                                                 - - tcacgcttgg tcctgtactt gaaaaaggga aagttggagc acatggtgta ag -            #tttcggtg   3180                                                                 - - tcagtgaaca atatgaagaa ttaaaaagta tgttaggtac gtggagtgat ac -            #aaatgatg   3240                                                                 - - attctgtgag agcgaatcgt ccgcgtattg atacagcacg taatgtagca ga -            #tgcaatac   3300                                                                 - - taagtatttc atctgctacg aatggtaaat tatcacaaaa atcatatgaa ga -            #tcttgaag   3360                                                                 - - aacaaactgg aatgccgtta aaagatattt ctagcgaacg tgctgctgag aa -            #aatttcgt   3420                                                                 - - ttttaaatat aacttcacaa ccacgagaag taataccgac agcagtattc cc -            #aggttcaa   3480                                                                 - - ataaacaagg tcgacgatat tcaccattta caacgaatat agaacgtcta gt -            #acctttta   3540                                                                 - - gaacattaac aggacgtcaa agttattatg tggatcacga agttttccaa ca -            #atttgggg   3600                                                                 - - agagcttacc agtatataaa ccgacattgc cgccaatggt atttgggaat ag -            #agataaga   3660                                                                 - - aaattaaagg tggtacagat gctttggtac tgcgttattt aacgcctcat gg -            #aaaatgga   3720                                                                 - - atatacactc aatgtatcaa gataataagc atatgttgac actatttaga gg -            #tggtccac   3780                                                                 - - cggtttggat atcaaatgaa gatgctgaaa aacacgatat ccaagataat ga -            #ttggctag   3840                                                                 - - aagtgtataa ccgtaatggt gttgtaacgg caagagcagt tatttcgcat cg -            #tatgccta   3900                                                                 - - aaggtacaat gtttatgtat catgcacaag ataaacatat tcaaacgcct gg -            #gtcagaaa   3960                                                                 - - ttacagatac acgtggtggt tcacacaacg cgccgactag aatccatttg aa -            #accaacac   4020                                                                 - - aactagtcgg aggatacgca caaattagtt atcactttaa ttattatgga cc -            #aattggga   4080                                                                 - - accaaaggga tttatatgta gcagttagaa agatgaagga ggttaattgg ct -            #tgaagatt   4140                                                                 - - aaagcgcaag ttgcgatggt attaaattta gataaatgca taggatgcca ta -            #cgtgtagt   4200                                                                 - - gtgacatgta aaaacacttg gacaaatcgt ccaggtgctg agtacatgtg gt -            #tcaataac   4260                                                                 - - gtagaaacga agccaggtgt agggtatccg aaacgttggg aagaccaaga ac -            #actacaaa   4320                                                                 - - ggtggttggg                - #                  - #                      - #      4330                                                                  - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 1229                                                            <212> TYPE: PRT                                                               <213> ORGANISM: Staphylococcus aureus                                          - - <400> SEQUENCE: 2                                                         - - Met Gly Lys Phe Gly Leu Asn Phe Phe Lys Pr - #o Thr Glu Lys Phe Asn       1               5  - #                10  - #                15               - - Gly Asn Trp Ser Ile Leu Glu Ser Lys Ser Ar - #g Glu Trp Glu Lys Met                  20      - #            25      - #            30                   - - Tyr Arg Glu Arg Trp Ser His Asp Lys Glu Va - #l Arg Thr Thr His Gly              35          - #        40          - #        45                       - - Val Asn Cys Thr Gly Ser Cys Ser Trp Lys Va - #l Phe Val Lys Asn Gly          50              - #    55              - #    60                           - - Val Ile Asn Trp Glu Asn Gln Gln Thr Asp Ty - #r Pro Ser Cys Gly Pro      65                  - #70                  - #75                  - #80        - - Asp Met Pro Glu Tyr Glu Pro Arg Gly Cys Pr - #o Arg Gly Ala Ser Phe                      85  - #                90  - #                95               - - Ser Trp Tyr Glu Tyr Ser Pro Leu Arg Ile Ly - #s Tyr Pro Tyr Ile Arg                  100      - #           105      - #           110                  - - Gly Lys Leu Trp Asp Leu Trp Thr Glu Ala Le - #u Glu Glu Asn Tyr Gly              115          - #       120          - #       125                      - - Asn Arg Val Ala Ala Trp Ala Ser Ile Val Gl - #u Asn Glu Asp Lys Ala          130              - #   135              - #   140                          - - Lys Gln Tyr Lys Gln Ala Arg Gly Met Gly Gl - #y His Val Arg Ser Asn      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Trp Lys Asp Val Thr Glu Ile Ile Ala Ala Gl - #n Leu Leu Tyr Thr        Ile                                                                                             165  - #               170  - #               175             - - Lys Lys Tyr Gly Pro Asp Arg Ile Ala Gly Ph - #e Thr Pro Ile Pro Ala                  180      - #           185      - #           190                  - - Met Ser Met Ile Ser Tyr Ala Ala Gly Ala Ar - #g Phe Ile Asn Leu Leu              195          - #       200          - #       205                      - - Gly Gly Glu Met Leu Ser Phe Tyr Asp Trp Ty - #r Ala Asp Leu Pro Pro          210              - #   215              - #   220                          - - Ala Ser Pro Gln Ile Trp Gly Glu Gln Thr As - #p Val Pro Glu Ser Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Trp Tyr Asn Ala Ser Tyr Ile Ile Met Tr - #p Gly Ser Asn Val        Pro                                                                                             245  - #               250  - #               255             - - Leu Thr Arg Thr Pro Asp Ala His Phe Met Th - #r Glu Val Arg Tyr Lys                  260      - #           265      - #           270                  - - Gly Thr Lys Val Ile Ser Val Ala Pro Asp Ty - #r Ala Glu Asn Val Lys              275          - #       280          - #       285                      - - Phe Ala Asp Asn Trp Leu Ala Pro Asn Pro Gl - #y Ser Asp Ala Ala Ile          290              - #   295              - #   300                          - - Ala Gln Ala Met Thr His Val Ile Leu Gln Gl - #u His Tyr Val Asn Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Pro Asn Glu Arg Phe Ile Asn Tyr Ala Lys Gl - #n Tyr Thr Asp Met        Pro                                                                                             325  - #               330  - #               335             - - Phe Leu Ile Met Leu Asp Glu Asp Glu Asn Gl - #y Tyr Lys Ala Gly Arg                  340      - #           345      - #           350                  - - Phe Leu Arg Ala Ser Asp Leu Gly Gln Thr Th - #r Glu Gln Gly Glu Trp              355          - #       360          - #       365                      - - Lys Pro Val Ile His Asp Ala Ile Ser Asp Se - #r Leu Val Val Pro Asn          370              - #   375              - #   380                          - - Gly Thr Met Gly Gln Arg Trp Glu Glu Gly Ly - #s Lys Trp Asn Leu Lys      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Leu Glu Thr Glu Asp Gly Ser Lys Ile Asn Pr - #o Thr Leu Ser Met        Ala                                                                                             405  - #               410  - #               415             - - Glu Gly Gly Tyr Glu Leu Glu Thr Ile Gln Ph - #e Pro Tyr Phe Asp Ser                  420      - #           425      - #           430                  - - Asp Gly Asp Gly Ile Phe Asn Arg Pro Ile Pr - #o Thr Arg Gln Val Thr              435          - #       440          - #       445                      - - Leu Ala Asn Gly Asp Lys Val Arg Ile Ala Th - #r Ile Phe Asp Leu Met          450              - #   455              - #   460                          - - Ala Ser Gln Tyr Gly Val Arg Arg Phe Asp Hi - #s Lys Leu Glu Ser Lys      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Gly Tyr Asp Asp Ala Glu Ser Lys Tyr Thr Pr - #o Ala Trp Gln Glu        Ala                                                                                             485  - #               490  - #               495             - - Ile Ser Gly Val Lys Gln Ser Val Val Ile Gl - #n Val Ala Lys Glu Phe                  500      - #           505      - #           510                  - - Ala Gln Asn Ala Ile Asp Thr Glu Gly Arg Se - #r Met Ile Ile Met Gly              515          - #       520          - #       525                      - - Ala Gly Ile Asn His Trp Phe Asn Ser Asp Th - #r Ile Tyr Arg Ser Ile          530              - #   535              - #   540                          - - Leu Asn Leu Val Met Leu Cys Gly Cys Gln Gl - #y Val Asn Gly Gly Gly      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Trp Ala His Tyr Val Gly Gln Glu Lys Cys Ar - #g Pro Ile Glu Gly        Trp                                                                                             565  - #               570  - #               575             - - Ser Thr Val Ala Phe Ala Lys Asp Trp Gln Gl - #y Pro Pro Arg Leu Gln                  580      - #           585      - #           590                  - - Asn Gly Thr Ser Trp Phe Tyr Phe Ala Thr As - #p Gln Trp Lys Tyr Glu              595          - #       600          - #       605                      - - Glu Ser Asn Val Asp Arg Leu Lys Ser Pro Le - #u Ala Lys Thr Glu Asp          610              - #   615              - #   620                          - - Leu Lys His Gln His Pro Ala Asp Tyr Asn Va - #l Leu Ala Ala Arg Leu      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Gly Trp Leu Pro Ser Tyr Pro Gln Phe Asn Ly - #s Asn Ser Leu Leu        Phe                                                                                             645  - #               650  - #               655             - - Ala Glu Glu Ala Lys Asp Glu Gly Ile Glu Se - #r Asn Glu Ala Ile Leu                  660      - #           665      - #           670                  - - Lys Arg Ala Ile Asn Glu Val Lys Ser Lys Gl - #n Thr Gln Phe Ala Ile              675          - #       680          - #       685                      - - Glu Asp Pro Asp Leu Lys Lys Asn His Pro Ly - #s Ser Leu Phe Ile Trp          690              - #   695              - #   700                          - - Arg Ser Asn Leu Ile Ser Ser Ser Ala Lys Gl - #y Gln Glu Tyr Phe Met      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Lys His Leu Leu Gly Thr Lys Ser Gly Leu Le - #u Ala Thr Pro Asn        Glu                                                                                             725  - #               730  - #               735             - - Asp Glu Lys Pro Glu Glu Ile Thr Trp Arg Gl - #u Glu Thr Thr Gly Lys                  740      - #           745      - #           750                  - - Leu Asp Leu Val Val Ser Leu Asp Phe Arg Me - #t Thr Ala Thr Pro Leu              755          - #       760          - #       765                      - - Tyr Ser Asp Ile Val Leu Pro Ala Ala Thr Tr - #p Tyr Glu Lys His Asp          770              - #   775              - #   780                          - - Leu Ser Ser Thr Asp Met His Pro Tyr Val Hi - #s Pro Phe Asn Pro Ala      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Ile Asp Pro Leu Trp Glu Ser Arg Ser Asp Tr - #p Asp Ile Tyr Lys        Thr                                                                                             805  - #               810  - #               815             - - Leu Ala Lys Ala Phe Ser Glu Met Ala Lys As - #p Tyr Leu Pro Gly Thr                  820      - #           825      - #           830                  - - Phe Lys Asp Val Val Thr Thr Pro Leu Ser Hi - #s Asp Thr Lys Gln Glu              835          - #       840          - #       845                      - - Ile Ser Thr Pro Tyr Gly Val Val Lys Asp Tr - #p Ser Lys Gly Glu Ile          850              - #   855              - #   860                          - - Glu Ala Val Pro Gly Arg Thr Met Pro Asn Ph - #e Ala Ile Val Glu Arg      865                 8 - #70                 8 - #75                 8 -      #80                                                                              - - Asp Tyr Thr Lys Ile Tyr Asp Lys Tyr Val Th - #r Leu Gly Pro Val        Leu                                                                                             885  - #               890  - #               895             - - Glu Lys Gly Lys Val Gly Ala His Gly Val Se - #r Phe Gly Val Ser Glu                  900      - #           905      - #           910                  - - Gln Tyr Glu Glu Leu Lys Ser Met Leu Gly Th - #r Trp Ser Asp Thr Asn              915          - #       920          - #       925                      - - Asp Asp Ser Val Arg Ala Asn Arg Pro Arg Il - #e Asp Thr Ala Arg Asn          930              - #   935              - #   940                          - - Val Ala Asp Ala Ile Leu Ser Ile Ser Ser Al - #a Thr Asn Gly Lys Leu      945                 9 - #50                 9 - #55                 9 -      #60                                                                              - - Ser Gln Lys Ser Tyr Glu Asp Leu Glu Glu Gl - #n Thr Gly Met Pro        Leu                                                                                             965  - #               970  - #               975             - - Lys Asp Ile Ser Ser Glu Arg Ala Ala Glu Ly - #s Ile Ser Phe Leu Asn                  980      - #           985      - #           990                  - - Ile Thr Ser Gln Pro Arg Glu Val Ile Pro Th - #r Ala Val Phe Pro Gly              995          - #       1000          - #      1005                     - - Ser Asn Lys Gln Gly Arg Arg Tyr Ser Pro Ph - #e Thr Thr Asn Ile Glu          1010             - #   1015              - #  1020                         - - Arg Leu Val Pro Phe Arg Thr Leu Thr Gly Ar - #g Gln Ser Tyr Tyr Val      1025                1030 - #                1035 - #               1040        - - Asp His Glu Val Phe Gln Gln Phe Gly Glu Se - #r Leu Pro Val Tyr Lys                      1045 - #               1050  - #              1055             - - Pro Thr Leu Pro Pro Met Val Phe Gly Asn Ar - #g Asp Lys Lys Ile Lys                  1060     - #           1065      - #          1070                 - - Gly Gly Thr Asp Ala Leu Val Leu Arg Tyr Le - #u Thr Pro His Gly Lys              1075         - #       1080          - #      1085                     - - Trp Asn Ile His Ser Met Tyr Gln Asp Asn Ly - #s His Met Leu Thr Leu          1090             - #   1095              - #  1100                         - - Phe Arg Gly Gly Pro Pro Val Trp Ile Ser As - #n Glu Asp Ala Glu Lys      1105                1110 - #                1115 - #               1120        - - His Asp Ile Gln Asp Asn Asp Trp Leu Glu Va - #l Tyr Asn Arg Asn Gly                      1125 - #               1130  - #              1135             - - Val Val Thr Ala Arg Ala Val Ile Ser His Ar - #g Met Pro Lys Gly Thr                  1140     - #           1145      - #          1150                 - - Met Phe Met Tyr His Ala Gln Asp Lys His Il - #e Gln Thr Pro Gly Ser              1155         - #       1160          - #      1165                     - - Glu Ile Thr Asp Thr Arg Gly Gly Ser His As - #n Ala Pro Thr Arg Ile          1170             - #   1175              - #  1180                         - - His Leu Lys Pro Thr Gln Leu Val Gly Gly Ty - #r Ala Gln Ile Ser Tyr      1185                1190 - #                1195 - #               1200        - - His Phe Asn Tyr Tyr Gly Pro Ile Gly Asn Gl - #n Arg Asp Leu Tyr Val                      1205 - #               1210  - #              1215             - - Ala Val Arg Lys Met Lys Glu Val Asn Trp Le - #u Glu Asp                              1220     - #           1225                                      __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide segment comprising apolynucleotide sequence or the full complement of the entire length ofthe polynucleotide sequence, wherein the first polynucleotide sequenceencodes a polypeptide comprising the amino acid sequence set forth inSEQ ID NO:2.
 2. A vector comprising the isolated polynucleotide segmentof claim
 1. 3. An isolated host cell comprising the vector of claim 2.4. A process for producing a polypeptide comprising the step ofculturing the host cell of claim 3 under conditions sufficient for theproduction of the polypeptide, wherein the polypeptide is encoded by thepolynucleotide sequence.
 5. A polynucleotide which encodes a fusionpolypeptide and which includes the isolated polynucleotide segment ofclaim
 1. 6. An isolated polynucleotide segment comprising apolynucleotide sequence or the full complement of the entire length ofthe first polynucleotide sequence, wherein the polynucleotide sequenceencodes a polypeptide consisting of the amino acid sequence set forth inSEQ ID NO:2.
 7. A vector comprising the isolated polynucleotide segmentof claim
 6. 8. An isolated host cell comprising the vector of claim 7.9. A process for producing a polypeptide comprising the step ofculturing the host cell of claim 8 under conditions sufficient for theproduction of the polypeptide, wherein the polypeptide is encoded by thepolynucleotide sequence.